Tetraploid watermelons producing small fruits

ABSTRACT

Tetraploid watermelon inbreds are disclosed. The invention relates to the seeds and plants of the tetraploid watermelon inbreds, the methods of propagating the tetraploid inbreds through seeds and tissue culture. The invention also relates to methods of producing the triploid seedless watermelon seeds and plants by crossing the tetraploid inbreds with diploid watermelon inbreds, and to the triploid plants produced therefrom.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national phase application of Internationalapplication PCT/US03/14849, filed May 12, 2003, which claims priority toU.S. application Ser. No. 10/387,873, filed Mar. 13, 2003 now abandoned,which claims priority to claim U.S. application Ser. No. 10/145,980,filed May 14, 2002, now U.S. Pat. No. 6,747,191.

FIELD OF THE INVENTION

This invention is in the field of watermelon breeding, specificallyrelating to a tetraploid watermelon used to produce triploid seeds andplants for production of a substantially small sized seedless watermelonfruit.

BACKGROUND OF THE INVENTION

Watermelon is an important horticultural crop that accounts for 2% ofthe world area devoted to vegetable crops. There were 6,024,000 acres ofwatermelon grown in the world and 187,000 acres of watermelons grown inthe United States in 1997. Asia is by far the most important watermelonproduction site with 72% of the world area and 77% of the worldproduction (FAO Production Yearbook 51, 1998). The estimated annualworld watermelon value exceeded $7.6 billion when using the UnitedStates average price for 1995-1997. The United States watermelon cropamounted to over 41 million cwt, from over 174,000 harvested acres, anda farm value of over $266 million, accounted for 9.2% of the harvestedacres, 10.0% of the production, and 3.5% of the value of the UnitedStates fresh vegetable industry in 1999 (USDA Agricultural Statistics2001). California was the leading state in watermelon farm gate value,exceeded $72 million in 2000, due to high percentage of triploidseedless watermelon grown in California. Seedless watermelon receiveswell above the average price for seeded watermelons in the market.Triploid seedless watermelon also produces higher yields than thediploid seeded watermelons.

Triploid seedless watermelon is a true F1 hybrid between a tetraploidwatermelon, as the female parent, and a diploid watermelon, as the maleparent (Kihara, H. 1951, Triploid Watermelons, Proceedings of AmericanSociety for Horticultural Science, 58:217-230). Regular watermelons, theseeded diploid watermelons, have 22 chromosomes (2N=2X=22) in theirsomatic cells. The tetraploid watermelons have 44 chromosomes (2N=4X=44)in their somatic cells. When female flowers of tetraploid plants arecross-pollinated by the male flowers of normal diploid plants, the seedsproduced in the fruit of tetraploid plants are triploid seeds. Triploidseeds produce triploid plants. When the triploids plants are grown withthe normal diploid plants in the same field, the triploid plants producefruits that are seedless. The seedless condition in triploid watermelonis the result of the presence of three homologous sets of chromosome persomatic cell rather than the usual two. Cells with three sets ofhomologous chromosomes are said to be triploid and are designated as 3X.The triploid seedless watermelons have 33 chromosomes (2N=3X=33) intheir somatic cells. The inability of the triploid zygote to producenormal viable gametes (pollen and egg cells) causes the absence of seedsin triploid fruits. Typically, seedless watermelons contain small ediblewhite ovules, similar to those in immature cucumbers.

Triploid seedless watermelons have been commercially grown in the UnitedStates since the late 1980's. The popularity of seedless watermelon hasincreased since its commercial introduction in the United States. Mostof the watermelons produced in California in 2001 were triploid seedlesswatermelons. Triploid varieties produce higher yields than the diploidseeded varieties, due to more fruit per plant and longer harvest period.The triploid seedless watermelon receives premium prices because of thehigh quality flesh virtually free of seeds.

The present invention addresses the need for triploid watermelonsproducing smaller seedless fruits while maintaining high standards offruit taste and texture. However, when the size of the fruit is reduced,the proportion of the rind in the fruit increases, leaving only littlespace for the edible flesh. Thus, it is also an objective of the presentinvention to develop watermelons producing small seedless fruits withhigh flesh content. The present invention also addresses the need fortetraploid watermelon plants, which can be used as parents for triploidwatermelons producing such small seedless fruits.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides new and unique tetraploidwatermelons. The tetraploid watermelons of the present invention arepreferably tetraploid inbred lines and are particularly suitable asparent for triploid hybrids watermelons. The tetraploid watermelons ofthe present invention produce small fruits with a very thin rind. Thefruits of the tetraploid watermelons of the present invention havepreferably a high content of soluble solids. Importantly, the tetraploidwatermelons of the present invention are capable of transmitting thesedesirable characteristics to their triploid progeny when crossed withdiploid watermelons. Therefore, the present invention also disclosestriploid watermelons that produce small-sized fruits virtually free ofseeds and having a very thin rind. Such seedless watermelon fruits havethe advantage of having more edible flesh with excellent taste andtexture when compared to watermelons of comparable size, and aretherefore particularly attractive to the consumer.

In a preferred embodiment, a tetraploid watermelon of the presentinvention produces a fruit weighting about 1.5 kg to about 2.5 kg,preferably about 1.5 kg to about 2 kg, preferably about 2 kg. In anotherpreferred embodiment, the rind of the fruit of a tetraploid watermelonof the present invention is about 4 mm to about 10 mm thick, preferablyabout 4 mm to about 7 mm thick. In another preferred embodiment, atetraploid watermelon of the present invention has excellent fruit set.In another preferred embodiment, a tetraploid watermelon of the presentinvention has high sugar content. In another preferred embodiment, atetraploid watermelon plant according to the present invention producesseeds weighting about 20 g to about 35 g per 1000 seeds, preferablyabout 23 g to about 33 g per 1000 seeds, preferably about 31 g per 1000seeds, in another preferred embodiment about 25 g per 1000 seeds. Inanother preferred embodiment, a tetraploid watermelon plant according tothe present invention produces up to about 150 seeds per fruit,preferably about 50 to about 150 seeds per fruit, preferably about 70 toabout 150 seeds per fruit, more preferably about 80 to about 150 seedsper fruit. In another preferred embodiment, said plant produces about3-4 fruits per plant. In another preferred embodiment, the fruit of saidplant comprises about 12 to about 14% of soluble solids, preferablyabout 13%. In another preferred embodiment, the fruit of said plantmatures about 24 to about 27 days after flowering.

In another preferred embodiment, a tetraploid watermelon of the presentinvention includes one or more of the following characteristics:

a) produces a fruit that is significantly smaller than the fruit ofconventional tetraploids, approximately ⅓ the size of conventionaltetraploids,

b) displays exceptional fruit set ability,

c) produces 3-4 fruits per plant in a greenhouse setting,

d) matures 7 to 10 days earlier than regular tetraploids as measuredfrom flower to fruit maturity,

e) the seed is much smaller in size than the seed of conventionaltetraploids,

f) the fruit rind is about ½ to ⅓ of the regular tetraploids,

g) the seed yield is significantly better than most of the tetraploidscreated with the same technique.

In yet another preferred embodiment, the present invention relates to anew and unique inbred tetraploid watermelon line, designated 90-4194 andincluding the characteristic above.

In another preferred embodiment, when a tetraploid watermelon of thepresent invention is used a parent for production of a triploidwatermelon, said triploid watermelon produces a small seedless fruithaving a very thin rind. Preferably, the seedless fruits weights about 2kg to about 3 kg. In another preferred embodiment, the rind of theseedless fruits is about 4 mm to about 13 mm thick, preferably about 7mm to about 11 mm thick.

In another preferred embodiment, when a tetraploid watermelon of thepresent invention is used a parent for production of a triploidwatermelon, said triploid watermelon including one or more of thefollowing characteristics:

-   -   a) small fruit size,    -   b) more fruit per plant,    -   c) fruits having higher sugar content,    -   d) thin rind, usually ½ or ⅓ the thickness of conventional        triploids,    -   e) the triploid fruits maturing about 7-10 days earlier than        currently available triploid seedless watermelon fruit,    -   f) the triploids of the invention are more tolerant to hollow        heart compared to currently available triploid seedless        watermelon fruits,    -   g) excellent fruit set ability,    -   h) soluble solid content above about 11%, preferably about 11%        to about 13%.

The present invention also includes the seeds of a tetraploid watermelonline of the present invention. The present invention also includespollen, ovule, fruit of a tetraploid watermelon of the presentinvention, and methods of producing hybrid triploid watermelon seedsusing a tetraploid watermelon line of the present invention. Thisinvention further includes triploid hybrid watermelon seeds produced bycrossing a tetraploid watermelon line of the present invention with adiploid watermelon line, and plants obtained from such seeds, includingfruits thereof.

The present invention further includes methods of producing seeds havingall the genetics of a tetraploid watermelon line of the presentinvention, methods of developing new inbred tetraploid lines using atetraploid watermelon line of the present invention, and methods forproducing a tetraploid watermelon line using a diploid watermelon line.

The present invention further provides:

A method of producing triploid hybrid watermelon seed by crossing atetraploid watermelon plant of the present invention with a diploidwatermelon plant. Preferably, said tetraploid plant is the femaleparent. Preferably, said diploid plant is the male parent.

A triploid hybrid watermelon seed produced by a method above.

The present invention further provides:

A method to produce triploid hybrid watermelon seed comprising the stepsof:

-   -   a) planting in pollinating proximity seed of a tetraploid        watermelon plant according to the present invention and seed of        a diploid watermelon plant;    -   b) planting seed of said diploid watermelon plant 7 to 10 days        earlier than seed of said tetraploid watermelon plant;    -   c) cultivating watermelon plants resulting from said seeds until        said plants bear second female flowers;    -   d) covering the female flowers of said tetraploid watermelon        plant on the afternoon immediately prior to the day said female        flowers open;    -   e) collecting the male flowers of said diploid watermelon plant        in early morning on the day said female flowers open;    -   f) pollinating said female flowers with said male flowers        collected in step e); and    -   g) harvesting seeds produced from said female flowers pollinated        in step f).

The present invention further provides:

A method to produce triploid watermelon seed according comprising thesteps of:

-   -   a) planting seed of a tetraploid watermelon plant according to        the present invention and seed of a diploid watermelon plant in        a field in a pattern of 2 rows of tetraploid watermelon plants        to 1 row diploid watermelon plants;    -   b) pruning said tetraploid watermelon plant to approximately 2-3        branches per plant;    -   c) emasculating substantially all the male flowers and male        flower buds on the plants of said tetraploid watermelon plant;    -   d) allowing pollination of the female flowers of said tetraploid        watermelon plant with the pollen of said diploid watermelon        plant;    -   e) marking the female flowers pollinated during the emasculating        period; and    -   f) harvesting the seed from the marked fruit of the tetraploid        watermelon plant.

The present invention further provides:

A method of producing tetraploid watermelon seed comprising: a) growinga tetraploid watermelon plant according to the present invention; b)allowing self-pollination of said tetraploid watermelon plant; c)obtaining a fruit from a plant of step b); d) extracting tetraploidwatermelon seed from said fruit. Preferably, the method furthercomprises washing and drying said seed.

The present invention further provides:

A method of developing a tetraploid watermelon line in a watermelonplant breeding program using plant breeding techniques, which includeemploying a watermelon plant, or its parts, as a source of plantbreeding material, comprising: obtaining the watermelon plant, or itsparts, of the present invention as a source of breeding material.

The present invention further provides:

A method for producing a tetraploid watermelon plant comprising:

-   -   a) obtaining a diploid watermelon line producing a small-sized        fruit with a thin rind,    -   b) subjecting plants of said diploid watermelon line to        tetraploid conversion to produce tetraploid watermelon plants,    -   c) selecting a tetraploid watermelon plant obtained in step b),        wherein said tetraploid watermelon plant produces a fruit        weighting about 1.5 kg to about 2.5 kg and having a rind with a        thickness of about 4 mm to about 10 mm.

In a preferred embodiment, the tetraploid watermelon produces a fruitweighting about 1.5 kg to about 2 kg, preferably about 2 kg. In anotherpreferred embodiment, the rind of the fruit of a tetraploid watermelonof the present invention is about 4 mm to about 7 mm thick. In anotherpreferred embodiment, the tetraploid watermelon line produces seedsweighting about 20 g to about 35 g per 1000 seeds. In another preferredembodiment, the tetraploid watermelon line produces up to about 150seeds per fruit, preferably about 50 to about 150 seeds per fruit,preferably about 70 to about 150 seeds per fruit, more preferably about80 to about 150 seeds per fruit. In another preferred embodiment, thetetraploid watermelon line produces about 3-4 fruits per plant. Inanother preferred embodiment, the fruit of said tetraploid watermelonline comprises about 12 to about 14% of soluble solids. In anotherpreferred embodiment, the fruit of said tetraploid watermelon linematures about 24 to about 27 days after flowering. In another preferredembodiment, the watermelon tetraploid line comprises any one of thecharacteristics disclosed herein.

In another preferred embodiment, said diploid watermelon line is adiploid inbred watermelon line, preferably diploid inbred watermelonline HD or a diploid inbred watermelon line having the characteristicsof diploid inbred watermelon line HD. In another preferred embodiment,the diploid line is converted to a tetraploid line using an oryzalinprotocol.

In another preferred embodiment, the method further comprisesself-pollinating a tetraploid watermelon plant selected in step c)above. In another preferred embodiment, the method further comprisesself-pollinating a tetraploid watermelon plant selected in step c) aboveuntil no variation is observed in the progeny.

The present invention further provides:

A tetraploid watermelon line obtainable by a method disclosedimmediately above, including pollen, ovule, fruit of said tetraploidwatermelon line.

DETAILED DESCRIPTION OF THE INVENTION

In commercial production of triploid watermelon seed, tetraploid anddiploid parental lines are planted in the same field. Cross-pollinationbetween the tetraploid line, the female parental line, and the diploidline, the male parental line, are accomplished by either hand or beepollination. Triploid watermelon seeds are produced only in melons oftetraploid plants that are fertilized with pollen of diploid plants. Allcommercially grown seeded watermelons are diploid; therefore, there aremany diploid lines for use as diploid parents. The major limitation todevelopment of seedless watermelon varieties lies in the availability ofuseful tetraploid parental lines. In general, it is difficult to predictthe performance of a tetraploid watermelon from based on thecharacteristics of the diploid. A good diploid watermelon does notnecessarily produce a good tetraploid. Morever, a tetraploid with goodhorticultural traits does not necessarily produce good triploidwatermelon hybrids.

Tetraploid watermelon lines are developed by doubling the chromosomes ofregular diploid watermelon lines. Chromosome doubling was firstaccomplished with the toxic alkaloid colchicine by applying colchicineto the growing point of new emerged watermelon seedlings. Tissue culturemethods have also been developed by Zhang, X. P., B. B. Rhodes, H. T.Skorupska, W. C. Bridges, 1995, Generating Tetraploid Watermelon UsingColchicine in Vitro, G. Lester & J. Dunlap et al. (eds.), Cucurbitaceae'94: 134-139. Dinitroanilines have been used to double chromosomenumbers, and their effectiveness has previously been compared with cropsother than watermelon. Li et al (Li, Ying, J. F. Whitesides, B. Rhodes,1999, In vitro generation of tetraploid watermelon with two differentdinitroanilines and colchicines, Cucurbit Genetics Cooperative Rpt22:38-40) compared in vitro chromosome doubling effectiveness usingcolchicine and the dinitroanilines, ethalfluralin(N-ethyl-N-2-methyl-2-propenyl)-2,6-dinitro-4-(trifluoromethyl)benzanine),and oryzalin (3,5-dinitro-N4,N4-dipropylsulfanilamide) and concludedthat either ethalfluralin or oryzalin was preferable to colchicine.

Several treatment methods are used to induce tetraploids from diploidsusing the chemicals mentioned above. One method is to treat the seedbefore sowing. The seed are soaked in clean water for 5-6 hrs and thenthe seed are soaked in either colchicine solution (0.2%) ordinitroanilines (e.g. 35 μM oryzalin) for 24 hrs. The seed are brieflyrinsed before sowing. Dry seed can also be directly soaked in thechemical solution without pre-soaking in the water. This treatment issimple to do and is a good method to use if one has no restriction ofseed supply. The treatment usually reduces the germination andemergence. The second method is to treat the new emerged seedling. Thediploid inbreds are sown in greenhouse in seedling flats. The soiltemperature is kept at 29-31° C. for rapid and uniform germination. Onedrop of colchicine (0.1%) or dinitroanilines (e.g. 35 μM oryzalin)solution is added to the shoot apex between the cotyledons as soon asthe seedling has emerged from soil. The colchicine solution is appliedto the growing point in the morning or evening for three consecutivedays. We get good chromosome doubling from one application of oryzalin.Another method is to treat the shoot apex of germinated seed after whichthe germinated seed is planted into soil. The seeds are germinated in anincubator at 30° C. When the radicals are about 2 cm long, the portionabove the hypocotyls of germinated seeds is immersed upside down intocolchicine (0.1%) or dinitroaniline solution (35 μM oryzalin) for 10-15hrs at 30° C. in an incubator. The treatment should be conducted in ahigh humidity chamber or box to assure that the radicals/roots are notdesiccated. The seeds are then washed and planted in the soil. The lasttwo methods, although more tedious to use, usually give better recoveryof tetraploid events as the root system is not affected by thetreatment.

The next step is to develop tetraploid lines from individual convertingevents. The selected tetraploid individuals based on morphologicalexpression are self-pollinated and the resulting seeds are planted inthe next generation as lines. These lines are again self-pollinated andcompared for fertility and horticultural traits. Only the desirablelines are selected if there is difference among these lines. Desirablelines may be bulk harvested if there is no variation within the line andamong selected lines. Further seed increases may be conducted in anisolation block. Mass selection may be conducted for this increase inthe isolation plot and thereafter. Fertility of the tetraploid may beimproved in subsequent generations.

The use of tissue culture to propagate tetraploid watermelon plants isfurther exemplified in Adelberg, J. W., B. B. Rhodes, Micropropagationfrom zygotic tissue of watermelon, C. E. Thomas (ed.) Proc. of theCucurbitaceae 89: Evaluation and enhancement of cucurbit germplasm,Charleston S.C., USA; and Zhang et al., Shoot regeneration from immaturecotyledon of watermelon, Cucurbit Genetics Coop. 17:111-115 (1994).

Crossing two different tetraploids and then going through recombinationbreeding can also result in new tetraploid lines. A longer breedingperiod is required to develop a stable tetraploid line using thisapproach. This is due to the larger number of combinations and the fewerseed that tetraploids produce. However, some breeders make good progressby taking this approach.

Because meiosis is sometimes irregular in autotetraploids, diploids andaneuploids do occur in their offspring. The leaves, flowers and pollengrains of tetraploids are morphologically distinct from diploids (Zhang,X. P., B. B. Rhodes, H. T. Skorupska, W. C. Bridges, 1995, GeneratingTetraploid Watermelon Using Colchicine in Vitro, G. Lester & J. Dunlapet al. (eds.), Cucurbitaceae' 94: 134-139). Tetraploids also have adifferent number of chloroplasts in the guard cells (Compton, M. E., D.J. Gray and G. W. Elmstrom. 1996, Identification of tetraploidregenerants from cotyledons of diploid watermelon cultures in vitro,Euphytica 87:165-172). These morphological traits can help breeder toeliminate the diploids and aneuploids occurring in the tetraploidpopulation during sexual propagation.

Triploid seeds are currently produced using two methods, thebee-pollination method and the hand-pollination method. In the UnitedStates, the bee-pollination method is used to produce triploidwatermelon seed. Almost all of the United States triploid watermelonseed production is located in Northern California. The production fieldsare typically planted in a ratio of 2 rows of tetraploid female line and1 row of diploid male line. All the male flower buds are manuallyremoved from the female tetraploid plants. This process is known asde-budding. The female flowers are open-pollinated by bees. The fruitset during the de-budding period are marked and harvested for triploidhybrid seed. Male buds are manually removed from tetraploid female vinesthroughout the pollination season. If a male sterile tetraploid line isavailable, workers can easily remove the male fertile plants in thetetraploid female row with much less time and efforts. All the fruit seton the male-sterile tetraploid plants can be harvested for hybridtriploid seed. When the marked male-sterile system is used, seedproducer can insure that no female off-types exist in the femaletetraploid line and the hybrid triploid seed (Zhang, X. P. and B. B.Rhodes, 2000, Method using male sterility and a marker to produce hybridseeds and plants; U.S. Pat. No. 6,018,101).

Hand-pollination is mainly used to produce triploid watermelon seed inareas where isolation is not available and several triploid hybrids areproduced in the same field block. Inbred male parent line is sown 7-10days earlier than inbred female tetraploid parent line. The male parentis usually located outside of the crossing block. Approximately four toten tetraploid female plants per male plant are planted to insureadequate pollination. The male parent is carefully checked for itsuniformity before male flowers are collected. Any off-types that can berecognized based on plant morphology and ovary characteristics areremoved. Pollination starts when the second female flowers of thetetraploid female parent are ready to flower. The female flower buds ofthe tetraploid female parent line are identified and covered with papercups or small paper bags before they bloom the next morning. Maleflowers of the diploid male parent line are collected in the earlymorning before the visit of bees or other pollination insects to theflowers. The covered female flower buds are then uncovered andpollinated using the collected fresh male flowers. The pollinated femaleflowers are then recovered and marked. The open-pollinated fruits on thefemale parent plants are removed periodically to insure the developmentof hand-pollinated fruits. Male plants are removed from the field afterpollination is complete to insure that only fruit from female parentsare harvested.

Development of Tetraploid Lines of the Present Invention

A problem regularly observed with the conversion of diploid watermelonlines to tetraploid lines is that the fertility rate of the tetraploidline is dramatically reduced. This leads to a low fruit set and is oftencorrelated with late fruit setting and late maturity of the fruit.Consequently, seeds production of tetraploid watermelons is inefficientand expensive, and tetraploid watermelons are generally not good parentsfor triploid hybrid seeds production. An objective of the presentinvention is therefore to produce tetraploid watermelons overcomingthese fertility problems, while having the advantageous characteristicsdisclosed in the instant application and being able to transmit thesecharacteristics to their triploid progeny in full or at least in part.

Diploid inbred watermelon line HD, a proprietary Syngenta line used as aparental line for the production of diploid seeded watermelon hybrids,was identified as a promising candidate for tetraploid conversion.Diploid line HD produces small-sized seeded fruits with a thin rind, andhas excellent fruit set. Moreover, diploid line HD produces fruits withhigh sugar content and has a firm flesh. Diploid watermelon line HD wassubjected to tetraploid conversion.

Inbred 90-4194 was developed in northern California by convertingdiploid inbred HD to a tetraploid watermelon. The conversion fromdiploid (2X) to tetraploid (4X) was accomplished using an oryzalinprotocol comprising the following steps:

In November of 1999, seeds of HD were seeded in a 50-cell plasticseedling tray in the greenhouse. One drop of 35 μM oryzalin was added tothe meristem tip between 2 cotyledons each of the newly emergedseedlings. Treatment of all the seedlings with oryzalin was finishedabout 10 days after sowing.

Seedlings were watered and fertilized periodically.

In late December of 1999, putative tetraploids were transplanted into2-gallon pots filled with Pro-Mix BX soil-less soil in the greenhouse.During the course of plant development, diploid (not converted) plantsand branches were removed based on leaf morphology and male flowercharacteristics.

Following is the chronological order of development of tetraploidwatermelon lines, for example inbred line 90-4194:

Generation Season/Year Description

T₀ Spring 2000 At the seedling transplant stage, 72 putative tetraploidswere transplanted into 2-gallon pots in greenhouse. Non-converted plantsand branches were identified based on leaf morphology and male flowercharacteristics, and were removed. About 20 tetraploids were identifiedand only the female flowers from true tetraploid plant/branches wereself-pollinated. At full fruit maturity, fruit with large blossom endscars (2-3 times that of its diploid version) were harvested andexamined for fertility as suggested by number of seed per fruit. Fourindividual selections 4XHD-1, -2, -3, and -4 and one bulk selection4XHD-B were made to plant the T₁ generation.T₁ Summer 2000 4XHD-1 and 4XHD-2 were planted in the greenhouse forfurther selection and seed increase. 4XHD-3 and 4XHD-4 were planted inthe field for field observation and seed increase. 4XHD-B was planted inthe crossing block in the field to make triploid hybrids. 4XHD-2 was notas good as 4XHD-1 at the seedling stage and was discarded. 42 plants of4XHD-1 were grown to maturity in the greenhouse. All the seeds of 4XHD-1were bulk-harvested and labeled as 4XHD-1-B as no variation was observedin this line. No variation was observed within and between 4XHD-3 and4XHD-4. Therefore seeds were also bulk-harvested and labeled as4XHD-3/4.

Fall 2000 Five triploid hybrids derived from 4XHD-B were evaluated inFlorida. Three hybrids were unique and promising triploid hybrids.

T₂ Spring 2001 About 700 plants of 4XHD-1-B were planted in a plasticgreenhouse for generation advance and seed increase. Hand pollinationwas conducted. No variation was observed. All the fruits are uniform andtrue to type. Seeds were bulk-harvested and named as 90-4194.T₃ Summer 2001 About 3500 plants of 90-494 were transplanted to aone-acre isolation plot for stock seed increase using bee-pollination.1200 plants of 90-4194 were transplanted into 2 net covered cages forfoundation seed increase by hand pollination. No variation was observedfrom cage and field plantings. The breeding process is finished andseeds harvested serve as foundation and stock seed.

The unique characteristics of a tetraploid watermelon line of thepresent invention, for example inbred 90-4194, are described as follows.The values indicated below are for plants grown under summer conditionsin Northern California. Variations may be observed when the plants aregrown under substantially different conditions.

A tetraploid watermelon line of the present invention is a very earlymaturing tetraploid watermelon. For example, fruit of 90-4194 matured24-27 days after flowering under summer conditions in NorthernCalifornia. Other commercial tetraploids take at least 35 days under thesame conditions.

The fruit of a tetraploid watermelon line of the present invention isvery small, about 1.5 kg to about 2.5 kg, preferably about 1.5 kg toabout 2 kg, preferably about 2 kg, only approximately ¼-⅓ the size ofthe tetraploids commercially used. The fruit shape is round and skincolor is light green with green pencil lines. The flesh is firm and redwith refractometer % soluble solids (mostly sugars) of about 12% toabout 14%, preferably about 13%.

The seeds of a tetraploid watermelon line of the present invention aresmall, preferably about 20 g to about 35 g per 1000 seeds, preferablyabout 23 g to about 33 g per 1000 seeds, preferably about 31 g per 1000seeds, in another preferred embodiment about 25 g per 1000 seeds,approximately ½-⅓ the weight of commercially used tetraploids.

A tetraploid line of the present invention has a good seeds yield. Theseeds yield is high when the tetraploid line is self-pollinated or whenthe tetraploid line is pollinated by a diploid male parent to producetriploid seeds. In a preferred embodiment, a tetraploid line of thepresent invention produces up to about 150 seeds per fruit, preferablyabout 50 to about 150 seeds per fruit, preferably about 70 to about 150seeds per fruit, more preferably about 80 to about 150 seeds per fruit.In another preferred embodiment, a tetraploid line of the presentinvention produces an average of about 80 seeds per fruit. The seedcolor is medium brown.

The rind is very thin, about 4 mm to about 10 mm, preferably about 4 mmto about 7 mm, approximately ½-⅓ the thickness of commercially usedtetraploids. This thin rind splits easily under dry conditions withgreat relative humidity fluctuations.

A tetraploid line of the present invention has excellent fruit settingability under poor environmental conditions. When 90-4194 was grown inthe plastic greenhouses without supplement lights in the early spring of2001 at Syngenta's Woodland research station with other 23 diploid and 1tetraploid breeding lines, it was the only watermelon line settingfruits normally Most of the watermelon lines did not produce any fruitdue to poor growing conditions.

A tetraploid line of the present invention can produce multiple fruitsper plant. For example, one plant of 90-4194 can bear as many as 4fruits per plant. A tetraploid line of the present invention can alsoproduce fruit in a long period of time. For example, 90-4194 produced 2crops from one planting during stock seed increase in the summer of 2001Northern California. After harvest of first fruit set 90-4194 produced asecond fruit set with excellent seed yield.

A tetraploid line of the present invention produces triploid hybridswith fruit size about 3 kg or smaller, preferably about 2.5 kg orsmaller. All the traditional triploid hybrids have much bigger fruits,most are above 6 kg. Preferably, fruits of the triploid hybrids furthercomprise one of the following characteristics: small fruit size, morefruit per plant (preferably 3-4 fruits per plant), higher sugar content,thin rind (the rind of the seedless fruits is about 4 mm to about 13 mm,preferably about 7 mm to about 11 mm), usually ½ or ⅓ the thickness ofconventional triploids, the triploid fruit mature about 7-10 daysearlier than regular triploid seedless watermelon fruit, the triploidsdeveloped from the invention are more tolerant to hollow heart,excellent fruit set ability, soluble solid content above about 11%,preferably about 11% to about 13%.

A tetraploid watermelon inbred of the present invention can bemultiplied through vegetative propagation and sexual propagation. Thevegetative propagation can be done preferably via shoot proliferationand then rooting in tissue culture. The detailed methods were describedby Zhang et al. (Zhang, X. P., B. B. Rhodes, H. T. Skorupska, W. C.Bridges. 1995. Generating Tetraploid Watermelon Using Colchicine inVitro. G. Lester & J. Dunlap et al. (eds.), Cucurbitaceae' 94: 134-139).This method is briefly described as follows: A) Collect shoot-tips andaxillary buds from greenhouse, field or laboratory grown plants of90-4194. Rinse the shoot-tips and axillary buds under tape water for 30minutes to an hour. Sterilize the shoot tips and axillary buds in 10% ofhousehold bleach for 8-10 minutes. Rinse the shoot-tips and axillarybuds three times in sterilized distilled water. B) Culture theshoot-tips and axillary buds on Murashige and Skoog (MS) medium with 10μM BA for shoot proliferation. Make a subculture every 3-4 weeks. C)Root the proliferated shoot buds on MS medium with 5-10 μM IBA for about2 weeks. D) Acclimatize the rooted plantlets in a temperature andhumidity controlled greenhouse. E) Grow the micropropagated plants in anisolation plot and harvest the seed from these plants.

Tetraploid watermelons are usually sexually propagated through seed. Wehave successfully propagated inbred 90-4194 in the greenhouse, in a netcovered cage, and in the open fields. The seed increase field should beisolated from any other watermelon by at least 1.5 KM, if the seedincrease is conducted in the open field. Good pest management andcultural practices should be implemented. Higher (20-30% higher thanthose used for commercial hybrid fruit production) levels of phosphateand potassium fertilize are beneficial for producing high seed yield andgood seed quality. Calcium is supplemented for the fields low in calciumto minimize the fruit loss due to fruit splitting. Beehives are placedin the seed production fields to insure good pollination, the keybiological event for seed production. Fruit is preferably harvestedbefore fruit split and deterioration. The harvested fruit is then storedunder room temperature for a period of time before extracting seed fromfruit, to allow further embryo development and seed maturation in thefruit. The seed, after being extracted from the flesh, is thoroughlywashed and quickly dried using a forced-air dryer to best maintain theseed viability.

The primary use of tetraploid watermelon is to make triploid hybridwatermelon seeds and plants that produce seedless fruit. The tetraploidline is used as female parent to cross with the diploid watermelonlines, the male parent lines. The creation of a desirable triploidhybrid heavily relies on the performance, especially seed producibility,and the combining ability of the tetraploid parent. A tetraploidwatermelon inbred of the present invention, for example line 90-4194, isa novel tetraploid line for creating triploid seedless watermelonhybrids having excellent fruit quality, small fruit size (personal sizein the range of 2-3 KG), early maturity and excellent fruit settingability.

It is preferable to select the diploid male parents on the basis ofsmall fruit size, ideally less than 8 lbs, preferably less than 6 lbs.In addition, the skin color and stripe pattern that is desired for thetriploid watermelon is selected from the diploid male parent, as theskin color and stripe pattern (non) of a tetraploid of the presentinvention are recessive to all other skin color and stripe patterns.Diploid male parents are also selected for their small seed size toreduce the size of the white seed ovules in the triploid fruit.

Most tetraploids are very difficult or fail to produce triploid seedwhen they are grown in greenhouse. However, inbred 90-4194 producedtriploid seed with good seed yield in the greenhouse. Inbred 90-4194also produced triploid seed in the field. Tetraploid watermelon lines ofthe present invention have very good combining ability in creatingtriploid hybrids. For example, with limited exploration, 5 desirabletriploid hybrids were created by crossing inbred 90-4194 with differentdiploid male lines.

Several methods can be used to produce triploid seeds from a tetraploidinbred line of the present invention, once the proper combination isdetermined. Two commonly used methods are described here with respect toline 90-4194 as example. Variations to these methods can be madeaccording to actual production situation.

Hand-pollination method. This is the most often used method forproducing triploid seed from 90-4194. The inbred tetraploid femaleparent 90-4194 and the inbred diploid male parent line are planted inthe same field. The inbred male parent is planted 7-10 day earlier thanthe female parent 90-4194 to insure adequate pollen supply at thepollination time. The male parent and female parent 90-4194 are plantedin the ratio of 1 male parent to 4-10 female parents. The diploid maleparent may be planted at the top of the field for efficient male flowercollection during pollination. Pollination is started when the secondfemale flower on the tetraploid female parent 90-4194 is ready toflower. Female flower buds that are ready to open the next day areidentified, covered with paper cups or small paper bags that prevent beeor any other insect visit of the female flowers, and marked with anykind of material that can be easily seen the next morning. This processis best done in the afternoon. The male flowers of the diploid maleparent are collected in the early morning before they are open andvisited by pollinating insects. The covered female flowers of thetetraploid female parent, which have opened, are un-covered andpollinated with the collected fresh male flowers of the diploid maleparent, starting as soon as the male flower sheds pollen. The pollinatedfemale flowers are again covered after pollination to prevent bees andany other insects visit. The pollinated female flowers are also marked.Only the marked fruits are harvested for extracting triploid hybridseed.

Bee-pollination method. Using the bee-pollination method, the tetraploidfemale parent 90-4194 and the diploid male parent are usually planted ina ratio of 2 rows tetraploid parent to 1 row male parent. The femaletetraploid plants are pruned to 2-3 branches. All the male flower budson the female tetraploid parent plants are removed manually, (thede-budding process), during the pollination season on a daily basis.Beehives are placed in the field for transfer of pollen by bees from themale parent to the female flowers of the female parent. Fruits setduring this de-budding time are marked. Only the marked fruits areharvested for extracting hybrid triploid seed.

The fruit of inbred 90-4194 split easily when they reach maturity, dueto its extremely thin rind. This can be a serious problem when the seedproduction is conducted under conditions of dramatic humidityfluctuations. Thus, the fruit is harvested right before the splitting,and stored in shade or under vine for a period of 2-3 weeks. The rindbecomes durable when the fruit is removed from the vine, and the embryoscontinue to develop in the harvested fruit, resulting in better seedquality.

According to the invention, tetraploid inbreds are used as parentallines to develop new tetraploid lines. The unique desirable traits atetraploid watermelon line of the present invention, for example line90-4194, make it also very useful as a parental line in the developmentof new tetraploid inbreds. Such line can be used as either female ormale parent to cross with another inbred or hybrid tetraploid to developnew tetraploid inbreds.

Example 1 Description of the Tetraploid Inbred 90-4194

-   Fruit: round, small-   Area of best adaptation: most areas-   Emergence of anthesis: 5 days earlier than the commercial tetraploid    variety 90-4231-   Pollination to maturity: 7 days earlier than the commercial    tetraploid variety 90-4231-   Ploidy: tetraploid-   Cotyledon: flat-   Sex: monoecious-   Number of Main Stems: 3 at crown-   Number of flowers at first fruit set: 18 staminate-    4 pistillate-   Stem: round, pubescent, 7 mm diameter at second node-   Internode length 7 cm-   Flower at first fruit set: staminate 3 cm across-    pistillate 3 cm across-    color yellow-   Mature fruit size: round, 15 cm long, 15 cm diameter at midsection,    2 kg average weight, smooth, light green rind with pencil lines,    mottle/net-   Rind: brittle, 4 mm thick blossom end, 7 mm thick sides-   Flesh: crisp, fine with little fiber, medium red, 13% soluble solids    of juice, no hollow heart, placental separation, or transverse crack-   Seed: 7 mm long, 5 mm wide, 2 mm thick, index (length÷width×10) is    14, 31 gm per 1000 seed, 80 seed per fruit, dark brown-   Sunburn: resistant

In contrast, the commercial tetraploid line 90-4231 has a fruit size ofapproximately 6-7 kg, and expresses some hollow heart, placentalseparation, and transverse crack.

In summary, the uniqueness of the inbred tetraploid watermelon line90-4194 of the invention includes the following: a) The tetraploid90-4194 produces a fruit that is significantly smaller than the fruit ofconventional tetraploids, approximately ⅓ the size of conventionaltetraploids; b) The tetraploid 90-4194 displays exceptional fruit setability. In Honduras, the tetraploid 90-4194 is able to produce 3 crops(harvest of triploid seed from the tetraploid plant) from 1 planting,whereas conventional tetraploid plants allow for only 1 such harvest.Likewise, in California, Peru, and Chile, the 90-4194 of the inventionproduces 2 crops from 1 planting. c) The tetraploid 90-4194 produces 3-4fruits per plant in a greenhouse setting, whereas conventionaltetraploids typically produce on the average of less than 1 fruit perplant. d) The tetraploid 90-4194 matures 7 to 10 days earlier thanregular tetraploids as measured from flower to fruit maturity. e) Thetetraploid seed of 90-4194 of the invention is much smaller in size thanthe seed of conventional tetraploids. Twelve to 17 seeds of conventionaltetraploid watermelon make a gram. In contrast, 35 to 39 90-4194 seedsof current invention make a gram. f) The fruit rind of 90-4194 is about½ to ⅓ of the regular tetraploids. g) The seed yield of the 90-4194tetraploid plant is significantly better than most of the tetraploidscreated with the same technique. 90-4194 produces as many as 150triploid seed per fruit when pollinated by diploid male parent. Most ofthe recently developed tetraploid watermelons typically produce about 10to 75 triploid seed per fruit when pollinated by diploid male parents.

Example 2 Triploid Watermelon Fruits

The triploids watermelon fruit grown from triploid seed produced by atetraploid plant of the invention display commercially important andnovel traits.

For example, the uniqueness of the triploids produced by 90-4194according to the invention, compared to conventional seedlesswatermelon, includes a) small fruit size, b) more fruit per plant, c)higher sugar content (the SSC), (see Table 1 below), d) thin rind,usually ½ or ⅓ the thickness of conventional triploids, e) the triploidfruit mature about 7-10 days earlier than regular triploid seedlesswatermelon fruit, f) the triploids developed from the tetraploid 90-4194of the invention are more tolerant to hollow heart, a common defect oftriploid seedless watermelon. Hollow heart on triploid seedlesswatermelon RWT 8123 and RWT 8124 has not been observed. RWT 8123 and RWT8124 are triploids produced by the tetraploid 90-4194. and g) excellentfruit set ability.

Table 1 shows the average weight (Avg. Wt.), the number of fruit perplant (Frt/plant) and the amount of soluble solids (mostly sugar) in thefruit flesh (SSC %). Tri-X-313 is a standard sized commercial triploidseedless watermelon, using a conventional tetraploid watermelon as thefemale parent. RWT8123 and RWT8124 are triploid seedless watermelonsusing the tetraploid 90-4194 watermelon of the invention as the femaleparent. The male diploid parents of the Tri-X-313, RWT8123, and RWT8124lines were chosen for their significantly different phenotypes.

TABLE 1 Tri-X-313 RWT8123 RWT8124 Avg. Avg. Avg. Wt. Frt/ Wt. Frt/ Wt.Frt/ Spacing (lbs) plant SSC % Spacing (lbs) plant SSC % Spacing (lbs)plant SSC % 80″ × 36″ 12.2 1.7 11.7 80″ × 12″ 4.1 2.2 13.3 80″ × 18″ 5.43.1 13.7 80″ × 36″ 12.1 1.8 80″ × 24″ 4.5 3.5 80″ × 36″ 12.5 1.6 80″ ×36″ 4.5 4.5

Table 2 below shows that the fruit of RWT 8124 (see above) is much smallthan regular triploid watermelon and that the fruit set of RWT8124, asexpressed by number of fruit per plant, is much higher than that ofcommercially available seedless watermelons.

TABLE 2 Average Fruit Weight Average Number of Fruit Per TriploidVariety (lbs) Plant 3X Sangria 18.28 Palomar 15.22 1.95 RWT8126 17.06RWT8124 6.15 3.52 RWT8139 14.94 RWT8140 15.52 Shadow 15.35 Tri-X-31315.69 2.02

In accordance with the method of the invention, it is preferable toselect the diploid male parents on the basis of small fruit size,ideally less than 8 lbs. In addition, the skin color and stripe patternthat is desired for the triploid watermelon is selected from the diploidmale parent, as the skin color and stripe pattern (non) of 90-4194 arerecessive to all other skin color and stripe patterns. Diploid maleparents are also selected for their small seed size to reduce the sizeof the white seed ovules in the triploid fruit.

Although the foregoing invention has been described in some detail inthis document, it will be obvious that changes and modification may bepracticed within the scope of he invention, as limited only by the scopeof the appended claims.

DEPOSIT

Applicants have made a deposit of at least 2500 seeds of diploid inbredwatermelon line HD [HD(2X90-4194)] with the American Type CultureCollection (ATCC), Manassas, Va., 20110-2209 U.S.A., ATCC Deposit No:PTA-5146, on Apr. 22, 2003. This deposit of the diploid inbredwatermelon line HD will be maintained in the ATCC depository, which is apublic depository, for a period of 30 years, or 5 years after the mostrecent request, or for the effective life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Additionally, Applicants have satisfied all the requirements of 37C.F.R. §§1.801-1.809, including providing an indication of the viabilityof the sample. Applicants impose no restrictions on the availability ofthe deposited material from the ATCC; however, Applicants have noauthority to waive any restrictions imposed by law on the transfer ofbiological material or its transportation in commerce. Applicants do notwaive any infringement of its rights granted under this patent or underthe Plant Variety Protection Act (7 USC 2321 et seq.).

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding.However, it will be obvious that certain changes and modifications suchas single gene modifications and mutations, somaclonal variants, variantindividuals selected from large populations of the plants of the instantinbred and the like may be practiced within the scope of the invention,as limited only by the scope of the appended claims.

All references cited herein are incorporated by reference in theapplication in their entireties.

1. A method for producing a tetraploid watermelon plant said methodcomprising: a) obtaining a diploid watermelon line, wherein said diploidwatermelon line is diploid inbred watermelon line HD, representativeseed of which has been deposited under ATCC Accession No. PTA-5146, b)subjecting plants of said diploid watermelon line to tetraploidconversion to obtain tetraploid watermelon plants, and c) selecting atetraploid watermelon plant obtained in step b).
 2. The method accordingto claim 1, wherein said step b) comprises subjecting said plants to anoryzalin treatment.
 3. The method according to claim 1, furthercomprising: d) self-pollinating the tetraploid watermelon plant selectedin step c).
 4. The method according to claim 3, further comprising: e)self-pollinating the tetraploid watermelon plant selected in step d)until no variation is observed in the progeny.
 5. A method for producinga tetraploid watermelon plant said method comprising: a) obtaining adiploid watermelon line having all the morphological and physiologicalcharacteristics of inbred watermelon line HD, representative seed of HDhaving been deposited as ATCC Accession No. PTA-5146, b) subjectingplants of said diploid watermelon line to tetraploid conversion toobtain tetraploid watermelon plants, c) selecting a tetraploidwatermelon plant obtained in step b), wherein said tetraploid watermelonplant produces a fruit weighing about 1.5 kg to about 2.5 kg and havinga rind with a thickness of about 4 mm to about 10 mm.
 6. The methodaccording to claim 5, wherein said step b) comprises subjecting saidplants to an oryzalin treatment.
 7. The method according to claim 5,further comprising: d) self-pollinating a tetraploid watermelon plantselected in step c).
 8. The method according to claim 7, furthercomprising: d) self-pollinating a tetraploid watermelon plant selectedin step c) until no variation is observed in the progeny.